Phorbol ester stimulation of prostanoid synthesis by cultured astrocytes

Abstract

The role of protein kinase C in mediating the synthesis and release of various prostanoids (prostaglandins E2, I2, F2 alpha and thromboxane A2) from astroglial cells derived from neonatal rat cerebrum and maintained in primary culture was investigated using phorbol ester. Phorbol myristate acetate stimulated the release of arachidonic acid from prelabelled cells and all 4 prostanoids in a dose-dependent manner (EC50 = 300 nM). This effect was inhibited by the protein kinase inhibitor 1-(-5-isoquinolinylsulfonyl)-2-methylpiperizine (IC50 = 25 microM) and the phospholipase A2 inhibitor, mepacrine (IC50 = 5 microM). In addition, the stimulatory effect of the phorbol ester was not apparent in cells which had been depleted specifically of protein kinase C. In the presence of the calcium ionophore A23187, phorbol ester-stimulated prostanoid release was enhanced. In the absence of extracellular calcium, there was no prostanoid-stimulation by phorbol ester, but the calcium channel blocker verapamil did not mimic this effect. We conclude that stimulation of protein kinase C by phorbol ester elicits prostanoid synthesis and release by a process that involves calcium influx and the activation of phospholipase A2.